A highly desirable alternative or complementary approach to preventing sexually transmitted diseases (STDs) focuses on the development of non- toxic broad spectrum microbicides that are effective under the conditions found within the vaginal tract. Ideally, these agents would be effective against the transmission of both cell-associated and cell-free virus, and prevent infection of cell native to the genital tract as well as infiltrating cells of immune system origin. The primary objective of this Project is to examine the efficacy of N-9, C31G, alkyl sulfates, and related compounds (singly and in combination) with respect to prevention of HIV-1 infection within the context of an in vitro cell culture system as well as explanted and transplanted human vaginal tract tissues. Secondarily, our investigations will afford an excellent opportunity to better understand several aspects of HIV-1 transmission and inactive within the vaginal tract, including the roles of cell-free and cell-associated viral transmission; the identities of susceptible vaginal target cells that are initially infected by the viral inoculum; expression of cell surface receptors which facilitate infection; the effect of the vaginal milieu (pH, temperature, seminal fluid, menstrual blood, and donated or synthetic cervical secretions) on HIV-1 transmission and inactivation, and the potential for co-infected pathogens to potential HIV-1 infection of susceptible cells within the vaginal tract. The Specific Aims of this Project are to: (1) utilize an in vitro cell culture system in continued studies of inactivation of lymphotropic, monocytotropic, and dual tropic HIV-1 strains by N-9, C31G, and alkyl sulfate microbicidal formulations under conditions relevant to the vaginal environment; (2) examine the cytotoxicity and anti-HIV-1 activity of microbicidal formulations with respect to infection of continuous human vaginal epithelial cell lines and primary human vaginal and anal keratinocytes cultured in vitro; (3) continue to characterize HIV-1 infections of human vaginal and anal xenografts, with emphasis on identification of cellular targets, the impact of cell-free and cell-associated virus, the effect of HSV-2 and HPV infection on HIV-1 infection, and HIV-1 transmission to systematic human immune cell populations from infected vaginal xenografts carried in SCID-hu mice; and (4) examine the cytotoxicity and anti-HIV-1 activity of microbicidal formulations proven to be effective anti-HIV-1 compounds in Specific Aims 1 and 2 within the context of human vaginal and anal xenografts.